This invention relates generally to apparatus and method for microscopic examination of a predetermined object field or plane within biological tissue.
In examining microscopic specimens of some thickness, the desired image is often obscured by light scattered from within the volume of the sample itself. This is particularly true of specimens examined by incident or reflected light, that is, when the illumination and viewing are from the same side of the specimen. The light scattering within the volume of the specimen above and below the plane of interest is reduced if only a small region is illuminated. The scattered light is further reduced if the illuminating light directed onto the object field follows a different path through the scattering medium than does the imaging light propagating from the object field.
A specific problem is the examination of the endothelial cell layer on the inner surface of the cornea of the human eye. These cells are responsible for maintaining the proper water content of the cornea, to prevent swelling and opacification of the cornea. To examine these cells it has been found effective to illuminate a narrow strip of cells using half the aperture of a microscope objective, and to use the other half of the aperture for viewing the cell layer.
The problem addressed by the present invention is the relatively small area which can be viewed or photographed by this method. The strip is typically only about 100 .mu.m wide and perhaps 400 .mu.m long. Even within this narrow strip the image quality is not uniform, generally being partially obscured by scattered light on one side. If a larger area is to be studied, it must be photographed sequentially in strips, which are then placed together as a composite.
Previous solutions to this problem have included a synchronized translation of the tissue and of the recording film. This solution is suitable for study of excised tissue but is not satisfactory for the living eye. Another solution suitable for in vitro studies employs two dimensional scanning of a microscope objective. A third suggested solution has employed a rotating Nipkow disc. While there solutions have shown some success for in vitro studies of excised tissue, they have not been successful in studies on living human eyes. The principal reason is the fact that the living human eye is in nearly constant motion, with only short intervals of time between small rotational movements, called saccades. Therefore, any scanning of such a subject must be done in an elapsed time which is short compared to the time between saccades.
Further solutions to the small field problem were proposed by U.S. Pat. No. 3,547,512 to Baer. The Baer arrangement employs an assembly of two slits and a mirror which oscillate as a unit about a specific axis of rotation. The motion of each slit must be equal to or greater than the width of the image which is produced. Uniform illumination must be provided over an area equal to the area of the image which is produced. Another solution employs one or more pairs of appropriately spaced slits located on a disc which rotates in its plane. This also requires a fairly large moving element and uniform illumination over an area the size of the final image.